Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

Abstract

Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C 21 ) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C 21 to distal processing organelles is dependent upon the NH 2 -terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C 21 constructs containing polyalanine substitution for Glu 11 -Thr 18 , 13 PPDY 16 , or 14 P, 16 Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH 2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35 S-labeled proSP-C 21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys 6 , Lys 34 , or both) failed to inhibit trafficking of EGFP/proSP-C 21 . These results indicate that PPDYmediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport. Surfactant protein C (SP-C)3 is a hydrophobic lung-specific protein that co-isolates with the biophysically active phospholipid fraction of pulmonary surfactant (1). The 3.7-kDa alveolar form of SP-C (SP-C 3.7 or mature SP-C) is a 35-amino acid, valine-rich peptide that results from synthesis of a 197 amino acid precursor form (proSP-C 21 ). Within the endoplasmic reticulum (ER), proSP-C 21 exists as a type II bitopic transmembrane protein (N cyt /C lumen ), which remains integrally membrane-associated while it is extensively processed by four post-translational endoproteolytic cleavages that take place in post-Golgi compartments (2-6). The resulting mature protein is transferred into the lumen of lamellar bodies for secretion into the alveolar space together with surfactant phospholipid and SP-B (7). Thus, the complete biosynthesis of SP-C 3.7 is critically dependent upon oligomeric sorting and targeting of proSP-C 21 to subcellular processing compartments distal to the ER/Golgi.Using deletional mutagenesis, we had previously shown that the NH 2 -terminal propeptide flanking domain (NTP) of proSP-C, which is oriented into the cytosol, contained potential targeting motifs for post-Golgi trafficking (8, 9). The candidate region included the amino acid sequence Met 10 -Thr 18 and, when present, was sufficient to direct transf...

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

“…The uniqueness of this motif was revealed with the employment of a known targeting motif of SP-C, the PPxY Nedd 4-2 binding motif ( 19,35 ). The biosynthesis of SP-C through the regulated pathway of alveolar epithelial cells is well established ( 41,42 ).…”

“…Using deletion and site-directed alanine mutagenesis, the single targeting motif of SP-C was localized to a PPDY motif within the 35 amino acid cytosolic N-terminal domain of the proprotein. Alanine substitution mutation of this motif resulted in ER retention of the propeptide ( 19,35 ).…”

“…In addition to large domain signals with complex structural architecture, small-size motifs containing short spanning amino acid residues have been shown to serve as major determinants in directing the intracellular routes and localization of protein to their site of function or secretion. Synaptic vesicle-targeted proteins ( 15 ), insulin receptor sorting to lysosomes ( 16 ), surfactant protein C routing to the lamellar body of alveolar type II cells (17)(18)(19), and the adherens junction-targeted shrew-1 transmembrane proteins ( 20 ) all represent proteins containing small targeting motifs that play a role in guiding proteins through proper intracellular routes and/or to their fi nal destinations.…”

“…First, the entire 35 amino acid N-terminal domain of proSP-C was swapped with the 21 amino acid N-terminal domain of ABCA3. Second, the targeting motif of proSP-C (SPPDY) ( 19 ) was replaced with the ABCA3 N-terminal motif (SP-C SPPDY → LLLWKN ) ( Fig. 1 ).…”

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

“…Another observation is the presence of 2 different organelles in the cytoplasm of the A549 cell, not clearly visible on the image obtained with the confocal microscope (Fig.1B) but now visible with the x100 immersion objective. This observation is not surprising as the cytoplasm of the cells has a complex organisation and different structures can be found such as the actin or microtubules 38,39 , endoplasmic reticulum 39,40 , the mitochondria 41 or the Golgi apparatus 42,43 .…”